Nav1.7 as a chondrocyte regulator and therapeutic target for osteoarthritis

Mice

C57BL/6 and Agc1-creERT2 mice were obtained from The Jackson Laboratory. Nav1.8-cre;Nav1.7flox/flox mice were mated with transgenic mice expressing Agc1-creERT2 to obtain inducible Nav1.7-knockout mice in chondrocytes and both chondrocytes and DRGs. For activation of creERT2 in adult mice, 150 mg kg−1 body weight of tamoxifen (Sigma, T5648) in sunflower seed oil (Sigma, S5007) was injected intraperitoneally into 10-week-old mice once a day for 5 consecutive days. Littermate controls were used for all experiments. All animals were housed on a 12-h light:dark cycle with ad libitum access to food and water in a specific pathogen-free environment. Animals were maintained on a C57BL/6 J background, and age matched males typically at 12 weeks of age were used, unless otherwise specified in the figure legends. No statistical methods were used to predetermine sample size. All animal studies were performed in accordance with institutional guidelines and approved by the Institutional Animal Care and Use Committee of New York University Grossman School of Medicine.

To establish the surgically induced DMM55 model, after ketamine and xylazine anaesthesia, the medial meniscotibial ligament in the right knee was sectioned with a blade to destabilize the medial meniscus. The chemically induced MIA OA model was established unilaterally via intra-articular injection of 0.1 mg of MIA (Sigma, I2513) in 6 µl of 0.9% sterile saline with a 30-gauge needle after anaesthetization with ketamine and xylazine56. Mice with OA models were randomized to receive different treatment within a cage. PF-04856264 (Alomone labs, 1235397-05-3), at 30 µg per g body weight or 1.5 µg per g body weight was orally delivered or injected intra-articularly, respectively, daily over a 4-week period starting from the first day of MIA injection. PF-04856264 was intra-articular injected at 1.5 µg per g body weight or orally delivered at 30 µg per g body weight every other day starting from 4 weeks post DMM surgery for a total of 8 weeks. CBZ (Sigma, C4024) was delivered through oral gavage at 250 mg per kg body weight daily over a 4-week period starting from the first day of MIA injection. Additionally, doses of 10, 50 or 250 mg per kg body weight CBZ were administered daily via oral gavage over an 8-week span, commencing 4 weeks after DMM surgery. To determine whether blocking HSP70 and/or midkine affected Nav1.7 blocker PF-04856264’s protective effects against OA in vivo, mice received oral administration of PF-04856264 at a dosage of 30 µg per g body weight every day, and they were simultaneously subjected to intra-articular injections of VER 155008 (Sigma, SML0271) at 0.55 µg/g body weight, iMDK (Tocris, 5126) at 0.9 µg per g, or a combined application of VER 155008 and iMDK daily starting from 4 weeks after DMM surgery for a total of 8 weeks.

Behaviour tests

OA-associated pain was measured using the von Frey assay and the open field travel analysis57 three times before establishment of the OA model, and every week starting from 8 weeks after DMM surgery, or at day 2, 4, 8, 14, 20 and 28 post MIA injection. All the behavioural tests were conducted in a blinded manner and performed between the hours of 12:00 and 17:00. von Frey filaments (Stoelting) were applied with increasing force intensities on the plantar surface of the hindpaw of the mouse which is placed in an elevated Plexiglass chamber with a metal grid floor that gave access to the plantar surface of the paws to determine the tactile pain threshold as based on a previous publication57. Rapid withdrawal of the hindpaw was recorded as a positive response. Hind paws were subjected to 10 trials at a given intensity with a 30-s interval maintained between trials and the number of positive responses for each von Frey filament’s stimulus was recorded. Animals were considered to have reached tactile threshold when 5 out of 10 trials generated a positive response. For open field travel analysis, mice were placed individually in a square clear chamber (45 × 45 cm) and allowed to freely explore for 2 min under normal lighting. Movement and trajectories of the mice were videoed and analysed by a computerized system.

Human subjects research

Human subjects research was performed according to the Institutional Review Boards at New York University Medical Center (institutional review board (IRB) study number i11-01488 and i9018). Human OA cartilage samples were collected from patients receiving total knee joint replacement surgery for OA at New York University Langone Orthopaedic Hospital. Non-arthritic femoral condyle cartilage specimens were obtained from fresh osteochondral allografts discarded following donor plug collection during surgical osteochondral allograft implantation. Cartilage samples used are surgical discards, and no consent is required based on our approved IRB, as we do not collect patient information except age, sex and clinical diagnosis of the samples, such as Osteoarthritis. OA and non-arthritic cartilage specimens were stored in liquid nitrogen immediately after collection until protein or RNA extraction.

A total of 22 non-OA and 165 patients with symptomatic knee OA from the New York biomarker cohort58 were included in this study according to the American college of rheumatology (ACR) criteria. The demographic data are summarized in Supplementary Table 2. Informed consent was obtained from all participating subjects. The IRB of the New York University Grossman School of Medicine approved this study (no. i05-131).

OA synovial fluid and serum samples were collected as part of an observational study to determine factors influencing knee OA pain improvement with hyaluronic acid visco-supplementation59. The synovial fluid samples were collected without joint lavage, and the volume ranged from 0.5 to 30 ml. The cell-free synovial fluids were prepared and frozen (−80 °C) within 1 h of collection. Collection and storage of synovial fluids were approved (no. 13-01257) by the IRB of the NYU Grossman School of Medicine. The demographic data are summarized in Supplementary Table 3.

For the full-thickness cartilage explant assay, human tibia plateaus were obtained from 8 deidentified patients with OA undergoing total knee arthroplasty. For each individual patient with OA, 12 full-thickness cartilage explants were isolated from areas with various degrees of OA-related cartilage degeneration with a 3-mm biopsy punch and randomly distributed into three different groups and treated with 10 ng ml−1 IL-1β, 10 ng ml−1 IL-1β plus 25 nM ProTx II, or 10 ng ml−1 IL-1β plus 1 µM PF-04856264 in DMEM medium for 5 days. The supernatant was collected and spun at 200g at 4 °C, followed by ELISA assay.

Electrophysiology

Primary human chondrocytes from patients with OA were grown in 100 mm tissue culture dishes in growth medium DMEM (Gibco, 11995-065) supplemented with 10% FBS (Hyclone, SH30088.03) and 1× penicillin–streptomycin (Thermo Fisher, 15070063). Chondrocytes were passaged every 5–7 days at 75–80% confluency no more than 3 times. Cells were plated at passaging into 12-mm round glass poly-d-lysine/laminin-coated coverslips (Corning, 354087) in 24-well plate format according to the following protocol: growth medium was removed, chondrocytes were rinsed once with 5 ml Ca2+ and Mg2+-free DPBS (Gibco, 14190-144) and incubated for 3–5 min with 1.5 ml 0.25% Trypsin/EDTA (Corning, 10222017), then cells were gently lifted off the dish and pipette-triturated in 8.5 ml of growth medium. Twenty-five microlitres of homogenized chondrocytes suspension was diluted into 1 ml growth medium at each cover glass to reach optimal cell density and were maintained in growth medium for 3–6 days until electrophysiological recordings.

Currents were recorded in whole-cell voltage clamp by Axopatch 200B amplifier (Molecular Devices). Recordings were low-pass filtered at 2 kHz and acquired at 100 kHz by Digidata 1440 A DAC using Clampex 10.7 software (Molecular Devices). p/4 leak subtraction protocol and sweep-averaging were used to subtract uncompensated leak and capacitance currents and to enhance signal/noise ratio. Pipettes were pulled from glass capillaries (PG52165-4; WPI) and had resistance 2–3.5 MΩ when filled with intracellular solution (in mM): 140 CsF, 10 NaCl, 10 HEPES, 1 EGTA, 20 dextrose, pH 7.3 with CsOH (328 mOsm l−1 with sucrose). Extracellular solution contained (in mM): 145 NaCl, 4 KCl, 2 CaCl2, 2 MgCl2, 10 HEPES; 10 TEA-Cl, 10 Dextrose, pH 7.4 with NaOH (327 mOsm l−1). Solutions for sodium current isolation were from ref. 10. The liquid junction potential was not compensated. Recordings were made at room temperature. Data were analysed using pClamp 10.7 (Molecular Devices) and Origin 2022b (OriginLab) software.

RNA extraction from human cartilage

For RNA isolation, about 1 g of cartilage was pulverized in liquid nitrogen and homogenized in Trizol at a concentration of 1 g tissue per 10 ml Trizol (Invitrogen, 15596026), followed by incubation at 4 °C with rotating for 2 h. Samples were mixed with 0.2 volumes of chloroform, vortexed for 20 s, and centrifuged at 14,000 rpm for 20 min at 4 °C. The aqueous phase was collected and gently mixed with an equal volume of isopropanol, followed by centrifugation at 14,000 rpm for 20 min at 4 °C. The resulting pellet was suspended in 350 µl of RLT buffer and processed for cleanup using the RNeasy Mini Kit (Qiagen, 74104) following the manufacturer’s instructions.

RNA assay by RT–qPCR

Total RNA extracted from chondrocytes or human cartilage was reverse transcribed using the High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems, 4387406). RT–qPCR was performed in triplicate with SYGR Green (Applied Biosystems, A25780) using human or mouse primers to Acan, Col2, Mmp13, Adamts5, Cox2, Nos2 and Gapdh (Applied Biosystems Real-time PCR system). mRNA levels were normalized to Gapdh and reported as relative mRNA fold change.

Histology

Human cartilage or mouse knee joints were fixed in 4% paraformaldehyde for 24 h before decalcification in 10% w/v EDTA for 2 weeks before paraffin embedding. The paraffin blocks were sectioned at a thickness of 5 µm and serial sections were subjected to Safranin O or haematoxylin and eosin (H&E) staining. Cartilage destruction was graded on Safranin O-stained sections by blinded observers using the OARSI histology scoring system60 (grade 0–6). Osteophyte development61 (grade 0–3) was evaluated and the thickness of the subchondral bone plate62 was measured on Safranin O or H&E-stained sections. Synovitis (grade 0–9) was determined based on the synovial lining cell layer enlargement, resident cell density, and inflammatory infiltration on H&E-stained sections63. For immunohistochemical staining, deparaffinized and hydrated sections were incubated with 0.1% trypsin for 30 min at 37 °C, followed by 0.25 U ml−1 chondroitinase ABC (Sigma-Aldrich, C3667) and 1 U ml−1 hyaluronidase (Sigma-Aldrich, H3560) for 60 min at 37 °C, respectively. After blocking, the sections were incubated with antibodies against Nav1.7 (1:50, Alomone Labs, ASC-008), COL2 (Invitrogen, cat. no. MA5-12789), COMP fragment64 (1:200, affinity-purified monoclonal), aggrecan neoepitope (1:100, Millipore, AB8135) and MMP13 (1:200, Abcam, ab3208) overnight at 4 °C. Detection was performed using the Vectastain Elite ABC kit (Vector Laboratories, PK6100), and the positive signal was visualized with 0.5 mg ml−1 3,3-diaminobenzidine in 50 mM Tris-Cl substrate (Sigma-Aldrich, D12384) and then counterstained with 1% methyl green (Sigma-Aldrich, 67060). Images were acquired with a Zeiss microscope. Semi-quantification analysis of the density of immunohistochemical staining for Aggrecan neoepitope, COMP fragment and collagen X was performed by ImageJ, and the same signal threshold was used for each group of similar immunohistochemical images65.

Cell culture

Primary articular chondrocytes were isolated from the femoral condyles and tibial plateaus of Nav1.7flox and Nav1.7chondrocyte mice on postnatal day 6 (ref. 66). Chondrocytes were maintained as a monolayer in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% FBS, 50 U/ml penicillin, and 0.05 mg ml−1 streptomycin. Articular chondrocytes at culture day 2 were treated as indicated for each experiment.

Primary articular chondrocytes were isolated from the femoral condyles and tibial plateaus of Nav1.7flox and Nav1.7chondrocyte mice at 12 weeks after DMM surgery. Chondrocytes were maintained as a monolayer in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% FBS, 50 U ml−1 penicillin, and 0.05 mg ml−1 streptomycin. After 5 days, the conditioned medium was collected and spun at 200g at 4 °C, followed by ELISA assay.

Human C28I2 chondrocytes were grown in DMEM medium supplemented with 10% FBS, 50 U ml−1 penicillin, and 0.05 mg ml−1 streptomycin. To knockdown Nav1.7, cells were transfected with commercially available siRNA (S534077, A134907) using Lipofectamine (Invitrogen, 13778100) as instructed by manufacture’s protocol. To fraction condition medium, C28I2 cells were stimulated with or without 25 nM ProTx II or PF-04856264. In brief, C28I2 cells were cultured with DMEM medium supplemented with 10% FBS, 50 U ml−1 penicillin and 0.05 mg ml−1 streptomycin. When cells reached 80% confluency, the medium was changed to DMEM supplemented with ITS Liquid Media Supplement (Sigma-Aldrich, I3146) containing 25 nM ProTx II or PF-04856264 for 2 days. The conditioned medium was then collected. After centrifugation to get rid of the cell debris, the medium was then fractioned based on molecular weight to >100 kDa, 30–100 kDa, 10–30 kDa and <10 kDa using Amicon Ultra-2 Centrifugal Filter Units sequentially (Fisher Scientific, UFC210024, UFC203024 and UFC201024). PF-04856264 and ProTx II have molecular weight of 437.492 and 3,826.65 Da, respectively. To exclude the effects of PF-04856264 and ProTx II in conditioned medium, ProTx II was depleted using dialysis tubing that allows the removal of molecules with molecular weights between 3.5–5 kDa (Micro Float-A-Lyzer 3.5–5 kDa, F235053, Thomas Scientific), while PF-04856264 was simply removed through dialysis against the medium. Conditioned medium with molecular weight of 30–100 kDa and 10–30 kDa were then analysed by mass spectrometry, performed by NYU Proteomics Laboratory. All MS/MS spectra were collected using the following instrument parameters: resolution of 15,000, automatic gain control (AGC) target of 5e4, maximum ion time of 120 ms, one microscan, 2 m/z isolation window, fixed first mass of 150 m/z, and normalized collision energy (NCE) of 27. MS/MS spectra were searched against a UniProt Human database using Sequest within Proteome Discoverer 1.4.

Western blotting

Western blot analyses were conducted with protein lysates from primary human cartilage and C28I2 cells. To determine the membrane localization of Nav1.7 in chondrocytes, cytosolic and membrane fractions of human C28I2 cells were extracted with Men-PER Plus Membrane Protein Extraction Kit (Thermo Fisher Scientific, 89842) and subjected to western blotting analysis. The following primary antibodies were used: Nav1.7 (1:500, Alomone Labs, ASC-008), TNFR2 (1:1,000, ProteinTech, 19272-1-AP), and GAPDH (1:5,000, ProteinTech, 60004-1-Ig).

ELISA

The levels of HSP70 and midkine in human sera and synovial fluid from healthy individuals and patients with OA, in conditioned medium and cell lysates of human C28I2 cells treated with 25 nM ProTx II or 1 µM PF-04856264 were measured by ELISA according to the manufacture’s instructions (Abcam, ab133060, ab193761). Before ELISA analysis, human synovial fluids were digested with hyaluronidase at 1 unit per 100 µl synovial fluid for 1 h at 37 °C. The levels of PRG4 and MMP13 in supernatant of human OA cartilage explants were measured by ELISA according to the manufacturer’s instructions, respectively (R&D systems, DY9829-05; Abcam, ab100605). The levels of HSP70 and midkine in mouse sera and conditioned medium collected from primary chondrocytes were measured by ELISA according to the manufacture’s instructions (Abcam, ab133061, ab279416).

Na+ and Ca2+ fluorescence imaging

Human OA chondrocytes or C28I2 cells were seeded on 8-well chamber (Thermo Fisher, 154461) and loaded with 5 µM CoroNa Green (Invitrogen, C36676) or Fluo-8 (Abcam, ab112129) in Hanks’ Balanced Salt Solution for 45 min at 37 °C in the presence or absence of 25 nM ProTx II or 1 µM PF-04856264 with or without 0.5 µM KB-R7943 (Tocris, 1244). CoroNa Green and Fluo-8 were excited at 488 nm and fluorescence images (525–530 nm) were acquired with 25x water-dipping objective on Zeiss 880 confocal microscope every 2 s during the experiment. Na+ and Ca2+ transients in chondrocytes were induced by 100 nM ATP in the presence or absence of 25 nM ProTx II or 1 µM PF-04856264. Fluorescence was expressed as the ratio of cytosolic fluorescence and initial intensity (F/F0).

Measurement of intracellular Ca2+ with plate reader

Intracellular Ca2+ in human OA chondrocytes or C28I2 cells was measured using Fluo-8 Calcium Flux Assay Kit (Abcam, ab112129) according to the manufacturer’s instructions. In brief, cells seeded in black walled 96-well plate (Corning, 3904) were loaded with Fluo-8 in Hanks’ Balanced Salt Solution for 30 min at 37 °C and 30 min at room temperature in the presence or absence of 25 nM ProTx II or 1 µM PF-04856264. Ca2+ transients in chondrocytes were induced by 100 nM ATP in the presence or absence of 25 nM ProTx II or 1 µM PF-04856264. Fluorescence was measured in a fluorescent plate reader with an excitation wavelength of 490 nm and emission wavelength of 525 nm, and expressed as the ratio of cytosolic fluorescence and initial intensity (F/F0).

Statistical analysis

All data are presented as mean ± s.d., unless otherwise specified in the figure or table legends. The numbers of mice used per genotype are indicated in figure legends. Comparisons between the two groups were analysed using two-tailed unpaired Student’s t-test unless stated otherwise in the figure legends. ANOVA with post hoc Bonferroni test was used when comparing multiple groups as described in the figure legends. A value of P < 0.05 was considered statistically significant. Statistical analyses were performed using GraphPad Prism 9.

Reporting summary

Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.


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